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Journal of Bacteriology, June 1999, p. 3552-3561, Vol. 181, No. 11
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Role of Region C in Regulation of the Heat Shock Gene-Specific Sigma Factor of Escherichia coli, sigma 32

Florence Arsène,1 Toshifumi Tomoyasu,1 Axel Mogk,1 Christiane Schirra,2 Agnes Schulze-Specking,1 and Bernd Bukau1,*

Institut für Biochemie und Molekularbiologie, Universität Freiburg, D-79104 Freiburg,1 and ZMBH, Universität Heidelberg, INF 282, D-69120 Heidelberg,2 Germany

Received 28 January 1999/Accepted 17 March 1999

Expression of heat shock genes is controlled in Escherichia coli by the antagonistic action of the sigma 32 subunit of RNA polymerase and the DnaK chaperone system, which inactivates sigma 32 by stress-dependent association and mediates sigma 32 degradation by the FtsH protease. A stretch of 23 residues (R122 to Q144) conserved among sigma 32 homologs, termed region C, was proposed to play a role in sigma 32 degradation, and peptide analysis identified two potential DnaK binding sites central and peripheral to region C. Region C is thus a prime candidate for mediating stress control of sigma 32, a hypothesis that we tested in the present study. A peptide comprising the central DnaK binding site was an excellent substrate for FtsH, while a peptide comprising the peripheral DnaK binding site was a poor substrate. Replacement of a single hydrophobic residue in each DnaK binding site by negatively charged residues (I123D and F137E) strongly decreased the binding of the peptides to DnaK and the degradation by FtsH. However, introduction of these and additional region C alterations into the sigma 32 protein did not affect sigma 32 degradation in vivo and in vitro or DnaK binding in vitro. These findings do not support a role for region C in sigma 32 control by DnaK and FtsH. Instead, the sigma 32 mutants had reduced affinities for RNA polymerase and decreased transcriptional activities in vitro and in vivo. Furthermore, cysteines inserted into region C allowed cysteine-specific cross-linking of sigma 32 to RNA polymerase. Region C thus confers on sigma 32 a competitive advantage over other sigma  factors to bind RNA polymerase and thereby contributes to the rapidity of the heat shock response.

* Corresponding author. Mailing address: Institut für Biochemie und Molekularbiologie, Universität Freiburg, Hermann-Herder Str. 7, D-79104 Freiburg, Germany. Phone: 49-761 203 52 22. Fax: 49-761 203 52 57. E-mail: bukau{at}sun2.ruf.uni-freiburg.de.

Journal of Bacteriology, June 1999, p. 3552-3561, Vol. 181, No. 11
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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